Nogalamycin monoxygenase (NMO) is a member of a family of enzymes that catalyze a key step in the biosynthesis of tetracycline antibiotics used to treat, for example, breast cancer in humans, using molecular oxygen for substrate oxidation but without an apparent cofactor. As most monoxygenases and dioxygenases contain a transition metal center (Fe/Cu) or flavin, this begs the question how NMO catalyzes this unusual oxygen atom transfer reaction from molecular oxygen to substrate directly. We performed a detailed computational study on the mechanism and catalytic cycle of NMO using density functional theory and quantum mechanics/molecular mechanics on the full protein. We considered the substrate in various protonation states and its reaction with oxidant O-2 as well as O-2(-center dot) through either electron transfer, proton transfer, or hydrogen atom transfer. The lowest energy pathway for the models presented here is a reaction of the neutral substrate with a superoxo anion radical (O-2(-center dot)). In the absence of available free superoxo anions, however, the alternative neutral pathway between O-3(2) and the substrate may be accessible at room temperature, although the barrier is higher in energy by about 20 kcal mol(-1) and therefore the reaction will be much slower. In contrast to previous experimental findings for both the enzymatic and uncatalyzed reactions, the mechanisms with the substrate in its deprotonated state were found to be high in energy, and therefore mechanistic suggestions are proposed. A thermodynamic analysis shows that the substrate has a very weak C-H bond that can be activated by a weak oxidant, and hence, a metal cofactor may not be needed for oxidizing this particular substrate. Finally, site-directed mutations were studied where active-site Asn residues were replaced, and the function of these residues in guiding oxygen to the C-12-position of the substrate was highlighted. Overall, NMO shows a versatile reactivity pattern, where the substrate can be activated by several low-energy pathways with oxidants and substrates in various oxidation and protonation state.